consists Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO).  Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

Category: Chromatography Protocols

FPLC Protocol. The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one. Sir William Dunn School of Pathology, Oxford University. - [Read FPLC Protocol]

Category: Microscopy Protocols: Optical Microscopy Protocols

Near-field scanning optical microscopy can achieve spatial resolution performance beyond the classical diffraction limit by employing a sub-wavelength light source or detector positioned in close proximity to a specimen. Such a sub-wavelength source usually consists of an aperture at the end of a tapered probe, which functions basically as a wave guide. Includes info.: Fiber Probe Fabrication; Pulling Method; Meniscus Etching; Selective Etching; Apertureless and Alternative Probe Designs etc. - [Read Near-Field Scanning Optical Microscopy: NSOM Probes]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

The protocol consists of a method for the generation of cytoplasmic extracts from mammalian cells (in this case, 293T cells) without the disruption of polyribosomes, the separation of ribosomal components and polyribosomes by sucrose gradient centrifugation, the isolation of mRNA from these fractions, and detection of mRNA by Northern blot analysis. - [Read Northern Blot Analysis of mRNA From Mammalian Polyribosomes Protocol]

Category: Protein Protocols: Protein Extraction Protocols

Protocol for Protein Extraction Using Proteomics. Extraction of proteins from plant cells that are rich in compounds that interfere with the 2-Dimensional electrophoretic separation methods such as salts, organic acids, phenolics, pigments, terpenes, among others. A common protocol used in our lab for extraction proteins from plant tissues consists in the homogenization of mortar-grounded material in liquid nitrogen with an extraction buffer. - [Read Protocol for Protein Extraction Using Proteomics]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]